U.S. patent application number 17/085985 was filed with the patent office on 2021-06-03 for expandable arrays and methods of use.
The applicant listed for this patent is Rutgers, The State University of New Jersey. Invention is credited to Daehoon Han, Howon Lee, Hatem E. Sabaawy, Chen Yang.
Application Number | 20210162408 17/085985 |
Document ID | / |
Family ID | 1000005457858 |
Filed Date | 2021-06-03 |
United States Patent
Application |
20210162408 |
Kind Code |
A1 |
Sabaawy; Hatem E. ; et
al. |
June 3, 2021 |
Expandable Arrays and Methods of Use
Abstract
An expandable array and methods of maintaining a biological
sample within an expandable array are provided. The expandable
array includes a plurality of receptacles configured to receive a
biological sample and a plurality of beams comprising a
programmable material. Each beam of the plurality of beams is
located between and connects at least two receptacles. The
programmable material can be a shape-memory polymer or a
magnetoactive material that transitions the plurality of beams from
an extended state to a contracted state upon application of a
stimulus.
Inventors: |
Sabaawy; Hatem E.; (New
Brunswick, NJ) ; Lee; Howon; (Piscataway, NJ)
; Yang; Chen; (New Brunswick, NJ) ; Han;
Daehoon; (New Brunswick, NJ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Rutgers, The State University of New Jersey |
New Brunswick |
NJ |
US |
|
|
Family ID: |
1000005457858 |
Appl. No.: |
17/085985 |
Filed: |
October 30, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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PCT/US2019/029931 |
Apr 30, 2019 |
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17085985 |
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62664740 |
Apr 30, 2018 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01L 2300/18 20130101;
B01L 3/50855 20130101; B01L 2300/0636 20130101 |
International
Class: |
B01L 3/00 20060101
B01L003/00 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This invention was made with government support under Grant
Nos. 5R01CA226746 and P30CA072720, awarded by the National Cancer
Institute, and Grant No. 18.times.092, awarded by the National
Institutes of Health. The government has certain rights in the
invention.
Claims
1-43. (canceled)
44. A kit comprising: an expandable array comprising: a plurality
of receptacles configured to receive a biological sample, and a
plurality of beams, each beam of the plurality of beams disposed to
extend between and connect at least two receptacles, the plurality
of beams comprising a a programmable material configured to
transition each beam from an expanded state to a contracted state
upon application of a stimulus; one or more biomolecules; and cell
culture medium or ingredients for making a cell culture medium.
45. (canceled)
46. (canceled)
47. (canceled)
48. An expandable array, comprising: a plurality of receptacles
configured to receive a biological sample; and a plurality of
beams, each beam of the plurality of beams disposed to extend
between and connect at least two receptacles, the plurality of
beams comprising a programmable material configured to transition
each beam from an expanded state to a contracted state upon
application of a stimulus.
49. The expandable array of claim 48, wherein the programmable
material is a magnetoactive material and the stimulus is a magnetic
field.
50. The expandable array of claim 49, wherein the magnetoactive
material comprises a polymer material within which magnetic or
magnetizable particles are disposed.
51. The expandable array of claim 50, wherein the magnetic or
magnetizable particles comprise neodymium iron boron.
52. The expandable array of claim 50, wherein the polymer is an
elastomer.
53. The expandable array of claim 49, wherein each beam of the
plurality of beams comprises at least two sections of magnetoactive
material having opposite magnetic orientation.
54. The expandable array of claim 53, wherein each beam is
configured to fold at a transition between the at least two
sections.
55. The expandable array of claim 48, wherein at least a subset of
the plurality of beams are disposed to extend from a top edge of a
respective receptacle.
56. The expandable array of claim 48, wherein, in the expanded
state, each beam of the plurality of beams extends horizontally
between connected receptacles.
57. The expandable array of claim 48, wherein, in the contracted
state, each beam of the plurality of beams is folded vertically
between connected receptacles.
58. The expandable array of claim 48, wherein the array has a width
of about 20 mm to about 30 mm and a length of about 25 mm to about
35 mm when each beam is in the contracted state.
59. The expandable array of claim 48, further comprising a handle
located at a perimeter of the array.
60. The expandable array of claim 48, wherein each receptacle of
the plurality of receptacles comprises a mesh structure.
61. The expandable array of claim 60, wherein the mesh structure
comprises a pore size of about 2 .mu.m to about 10 .mu.m.
62. The expandable array of claim 48, wherein the plurality of
receptacles is arranged in an 8.times.12 array.
63. The expandable array of claim 48, wherein the plurality of
receptacles is arranged in a 4.times.6 array.
64. A method of maintaining a biological sample, comprising:
placing an expandable array comprising a plurality of receptacles
and a plurality of beams in a multiwell plate, each beam of the
plurality of beams disposed to extend between and connect at least
two receptacles and comprising a programmable material configured
to transition each beam from an expanded state to a contracted
state upon application of a stimulus; placing a biological sample
within at least a subset of the plurality of receptacles; removing
the expandable array containing the biological sample from the
multiwell plate; exposing the expandable array to the stimulus, the
plurality of beams responsively transitioning to a contracted
state, the biological sample being maintained within the array
during transition.
65. The method of claim 64, wherein exposing the expandable array
to a stimulus includes exposing the array to a magnetic field.
66. The method of claim 64, further comprising transferring the
expandable array containing the biological sample to a histology
cassette.
67. The method of claim 66, wherein a relative configuration of the
receptacles is maintained during transfer of the expandable array
from the multiwell plate to the histology cassette.
68. The method of claim 66, wherein the biological sample is
maintained within the receptacles during transfer of the expandable
array from the multiwell plate to the histology cassette.
69. The expandable array of claim 48, wherein the programmable
material is a shape-memory polymer.
70. The method of claim 64, wherein exposing the expandable array
to a stimulus includes exposing the array to a temperature change.
Description
RELATED APPLICATION
[0001] This application is a continuation-in-part application of
International App. No. PCT/US2019/029931, filed on Apr. 30, 2019,
which claims the benefit of U.S. Provisional Application No.
62/664,740, filed on Apr. 30, 2018. The entire teachings of the
above applications are incorporated herein by reference.
BACKGROUND
[0003] 3D printing (3DP) is a class of processes where successive
layers of material are aggregated incrementally to directly form a
three-dimensional (3D) object [1]. Several 3DP techniques have been
introduced over the past several decades [1,2]. While individual
processes differ depending on the material and machine technology
used, the 3DP processes that use a lithographic technique using a
digital light processing (DLP) technology have made great progress
in the past several years [3-5].
[0004] Projection micro-stereolithography (P.mu.SL) is a micro
additive manufacturing technique based on DLP that has been
developed and that can provide for high-resolution, rapid, and
scalable printing of 3D objects. P.mu.SL uses a spatial light
modulator, typically a digital micro-mirror device (DMD), as a
dynamically reconfigurable digital photomask. P.mu.SL is capable of
fabricating complex 3D micro-structures in a bottom-up,
layer-by-layer fashion.
SUMMARY
[0005] Expandable arrays that can be manufactured by 3DP
techniques, including, for example, by P.mu.SL, and methods of
maintaining biological samples in such expandable arrays are
provided. The arrays can provide for the streamlined processing of
biological samples from collection and/or cell culturing to
histological analysis.
[0006] An expandable array includes a plurality of receptacles
configured to receive a biological sample and a plurality of beams
comprising a shape-memory polymer. Each beam of the plurality of
beams is disposed to extend between and connect at least two
receptacles.
[0007] The receptacles of the array can also comprise a
shape-memory polymer. The shape-memory polymer can be a thermally
responsive polymer, for example, a polymer having a transition
temperature of greater than about 37.degree. C. and less than about
80.degree. C., or greater than about 37.degree. C. and less than
about 50.degree. C. The shape-memory polymer can be an
acrylate-based polymer or methacrylate-based polymer. Examples of
suitable shape-memory polymers include polyacrylic acid,
polyethylene glycol diacrylate, polyethylene glycol dimethacrylate,
diethylene glycol dimethacrylate, bisphenol A ethoxylate
dimethacrylate, tert-butyl acrylate, and n-Butyl methacrylate. The
shape-memory polymer can be compatible with P.mu.SL printing
methods.
[0008] The beams, or at least a subset of the beams, can be of an
expandable shape, including, for example, a serpentine shape, a
corrugated shape, a pleated shape, a helical shape, or a folded
shape. The beams can be disposed to extend between sides of the
receptacles, to extend from a top edge of the receptacles, to
extend from a bottom edge, or any combination thereof. Each beam of
the plurality of beams can be configured to be disposed in an
extended state and a contracted state. A distance between each of
the receptacles can be about 2 to about 10 times greater, or about
4 to about 5 times greater, when each beam is in the expanded state
than the contracted state. The beams can be configured to revert to
the contracted state from the extended state at a transition
temperature of the shape memory polymer. Each of the receptacles
can be connected to each neighboring receptacle by at least one
beam.
[0009] The expandable array can be configured to fit within a
histology cassette when the beams are in the contracted state. For
example, the array can have a width of about 20 mm to about 30 mm
and a length of about 25 mm to about 35 mm when each beam is in the
contracted state. In a particular example, the array can have a
width of about 24 mm and a length of about 30 mm when each beam is
in the contracted state. The expandable array can be further
configured to be received by a multiwell plate, such as a 96-well,
24-well, or 6-well plate, when the beams are in the expanded state.
For example, the plurality of receptacles can be arranged in an
8.times.12, 4.times.6, or 2.times.3 array.
[0010] The array can further include at least one handle located at
its perimeter, such as to provide for easy handling of the array
during transport of the array from a multiwell plate to a histology
cassette. If at least two handles are included in the array, each
handle can be located at an opposing side of a perimeter of the
array. Each handle can be connected to at least two receptacles,
although the handles may be connected to multiple receptacles, for
example, to provide for more secure handling of larger arrays.
[0011] Each receptacle can comprise a mesh structure, such as a
mesh structure configured to retain a biological sample. The mesh
structure can have a pore size of about 2 .mu.m to about 10 .mu.m.
The dimensions of each receptacle can vary. Each receptacle can
have a diameter configured to interface with or fit within a
diameter of a well. The diameter of each receptacle can be, for
example, of about 0.5 mm to about 2.5 mm, of about 0.5 mm to about
1.5 mm, or of about 1 mm. Similarly, each receptacle can have a
depth configured to interface with or fit within a well. Each
receptacle can have, for example, a depth of about 2 mm to about 15
mm, of about 5 mm to about 15 mm, of about 3 mm to about 5 mm, or
of about 11 mm. A height of a combined receptacle and connecting
beam(s) can be of about 5 mm to about 15 mm in a contracted state.
The wall thickness of each receptacle can be of about 50 .mu.m to
about 150 .mu.m, or of about 100 .mu.m.
[0012] A method of maintaining a biological sample includes placing
an expandable array in a multiwell plate and placing a biological
sample within at least a subset of the plurality of receptacles of
the array. The method further includes removing the expandable
array containing the biological sample from the multiwell plate and
exposing the expandable array to a stimulus. The plurality of beams
of the expandable array responsively transition to a contracted
state, with the biological sample being maintained within the array
during the transition.
[0013] The exposure of the expandable array to a stimulus can
include exposure to a temperature change, for example, an increase
in temperature, such as provided by a heat source. The method can
further include transferring the expandable array containing the
biological sample to a histology cassette. During transfer, a
relative configuration of the receptacles can be maintained and the
biological sample can be maintained within the respective
receptacles (e.g., in a same or similar orientation).
[0014] The placement of a biological sample within the
receptacle(s) of the array can include seeding a cell culture
within at least a subset of the receptacles. The biological sample
can be, for example, cells, simple spheroids, mixed spheroids, or
organoids. Alternatively, the biological sample can be a tissue
specimen.
[0015] A kit includes an expandable array, one or more biomolecules
and a cell culture medium or the ingredients for making a cell
culture medium. The biomolecules can be, for example, a growth
factor and/or an extracellular matrix component.
[0016] An expandable array, as described above, can include other
types of programmable materials, instead of or in addition to
shape-memory polymers. The programmable material can be configured
to transition each beam from an expanded state to a contracted
state upon application of a stimulus.
[0017] The programmable material can be a magnetoactive material,
and the stimulus can be a magnetic field. The magnetoactive
material can comprise a polymer material, such as an elastomer,
within which magnetic or magnetizable particles are disposed. The
magnetic or magnetizable particles can comprise a ferromagnetic or
ferrimagnetic material. For example, the magnetizable particles can
be particles of neodymium iron boron.
[0018] Each beam can include two or more sections of magnetoactive
material having opposite magnetic orientation, so as to provide for
a folding of the beam upon application of a magnetic field. The
beam can be configured to fold at a transition between the at least
two sections. Other beam arrangements can be as described above.
The receptacles and overall configuration of the expandable array
can include features as described above.
[0019] Methods of maintaining a biological sample, as described
above, can include exposing the expandable array to a stimulus that
is a magnetic field.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0021] The foregoing will be apparent from the following more
particular description of example embodiments, as illustrated in
the accompanying drawings in which like reference characters refer
to the same parts throughout the different views. The drawings are
not necessarily to scale, emphasis instead being placed upon
illustrating embodiments.
[0022] FIG. 1 is a schematic of an expandable array and methods of
making and using same.
[0023] FIG. 2A is a top view of an expandable array in a contracted
state. Small grid lines are 2 mm and large grid lines are 10
mm.
[0024] FIG. 2B is a top view of the expandable array of FIG. 2A in
an expanded state. Small grid lines are 2 mm and large grid lines
are 10 mm.
[0025] FIG. 2C is a side view of the expandable array of FIG. 2A.
Small grid lines are 2 mm and large grid lines are 10 mm.
[0026] FIG. 3A is perspective view of an expandable array in a
substantially expanded state.
[0027] FIG. 3B is a perspective view of the expandable array of
FIG. 3A during contraction.
[0028] FIG. 3C is a perspective view of the expandable array of
FIG. 3A after having further contracted.
[0029] FIG. 3D is a perspective view of the expandable array of
FIG. 3A in a substantially contracted state.
[0030] FIG. 4A is a graph of temperature dependent modulus change
for four shape-memory polymers (SMPs), each of a different mixing
ratio of benzyl methacrylate (BMA) and poly (ethylene glycol)
dimethacrylate (PEGDMA).
[0031] FIG. 4B is a graph of the shift of glass transition
temperature (T.sub.g) of the SMPs of FIG. 4A as measured by Dynamic
Mechanical Analysis (DMA).
[0032] FIG. 4C is a graph of glass transition temperatures
(T.sub.g) of four SMPs as a function of percentage of crosslinker.
In the legend, B stands for Benzyl methacrylate (BMA), P550 for
Poly (ethylene glycol) dimethacrylate (PEGDMA) with molecular
weight (Mw) 550, P750 for Poly (ethylene glycol) dimethacrylate
(PEGDMA) with Mw 750, BPA for Bisphenol A ethoxylate
dimethacrylate, and DEG for Di(ethylene glycol) dimethacrylate.
[0033] FIG. 5A is a schematic of another example of an expandable
array and example methods of making and using same.
[0034] FIG. 5B illustrates an example of a receptacle that includes
connecting beams in a contracted state.
[0035] FIG. 5C illustrates the receptacle of FIG. 5B with
connecting beams in an expanded. or extended, state.
[0036] FIG. 6A is a graph of storage modulus and loss modulus
versus temperature from a dynamic mechanical analysis (DMA) of
3D-printed and molded specimens of a shape memory polymer
(SMP).
[0037] FIG. 6B is a graph of failure strain versus temperature from
a tensile test of laser-cut molded specimens of the SMP.
[0038] FIG. 6C is a graph of stress, strain, and temperature over
time from a shape programming and shape recovery test of the
SMP.
[0039] FIG. 7A illustrates an example of a prototype receptacle in
contracted and extended states.
[0040] FIG. 7B illustrates schematic of a cassette configuration
and a well plate configuration for an array of the prototype
receptacles of FIG. 7A.
[0041] FIG. 7C illustrates ratio of original size of a prototype
receptacle to a measured size of the receptacle over time during a
heat recovery test of a prototype array according to FIG. 7B.
[0042] FIG. 8 illustrates a process for operation of an expandable
array.
[0043] FIG. 9A is a schematic illustrating an expandable array
mounted within a stretcher in an initial, contracted
configuration.
[0044] FIG. 9B is a schematic illustrating the expandable array and
stretcher of FIG. 9B in an expanded configuration.
[0045] FIG. 9C depicts an example array mounted within an example
stretcher in the configuration shown in FIG. 9A.
[0046] FIG. 9D depicts the example array and stretcher of FIG. 9C
in the expanded configuration shown in FIG. 9B.
[0047] FIG. 10A is a schematic illustrating a top-side view of a
plate fixture.
[0048] FIG. 10B is a schematic illustrating a side view of the
plate fixture of FIG. 10A.
[0049] FIG. 11A depicts a prototype array in an expanded state with
markers at bridge connections for shape recovery mapping.
[0050] FIG. 11B depicts the photographed markers of the prototype
array of FIG. 11A.
[0051] FIG. 11C depicts the detected markers of the prototype array
of FIGS. 11A and 11B.
[0052] FIG. 12A illustrates a test process for histological
processing with a prototype expandable array.
[0053] FIG. 12B illustrates Hematoxylin and Eosin (H&E) stained
microtome sections obtained from the histological processing shown
in FIG. 12A. The scale bar in 100 .mu.m.
[0054] FIG. 13 depicts cross-section and side-section photos of
prototype receptacles with and without cells.
[0055] FIG. 14A depicts bright field images of glioblastoma (GBM)
spheres derived from U87 cells in the presence of fetal bovine
serum (FBS) and grown with and without receptacle arrays.
[0056] FIG. 14B depicts bright field images of GBM organoids grown
in serum-free conditions with growth factors and derived from GBM
#50 cells, grown with and without receptacle arrays.
[0057] FIG. 14C depicts bright field images of GBM #50 cells grown
in Matrigel spheres, grown with and without receptacle arrays.
[0058] FIG. 15A is a graph of cell titer glo assay results versus
component biomaterials for testing to determine GBM cell viability
in receptacle arrays.
[0059] FIG. 15B is a low-power bright field image of control U87
spheres cultured for one week for the control of FIG. 15A, shown in
5.times. and 20.times. magnification.
[0060] FIG. 15C depicts low-power bright field images of U87
spheres cultured for one week with the biomaterials of FIG. 15A,
shown in 5.times. and 20.times. magnification.
[0061] FIG. 16A depicts images of sphere cultures with cut
baskets.
[0062] FIG. 16B depicts images of sphere cultures from samples of
individually examined basket biomaterials.
[0063] FIG. 16C depicts images of cell cultures without basket
arrays and with basket arrays soaked in a 100% acetone bath after
3D printing.
[0064] FIG. 16D depicts images of GBM organoid cultures without
acetone soak and with 12 h and 24 h acetone soak.
[0065] FIG. 16E depicts bright field images showing no GBM organoid
grown in a basket when an acetone bath was not used and GBM
organoid growth in a basket soaked in 100% acetone bath after 3D
printing.
[0066] FIG. 16F is a graph of the results of the acetone soak trial
depicted in FIG. 16D of Relative Light Units (RLU) versus control,
12 hour soak, and 24 hour soak.
[0067] FIG. 17 is a schematic of another example of an expandable
array and methods of using same.
[0068] FIG. 18A illustrates an example of a receptacle that
includes connecting beams in an expanded state.
[0069] FIG. 18B illustrates an example of receptacles as in FIG.
18A with connecting beams in a contracted state.
[0070] FIG. 19 is a schematic illustrating a method of programming
a magnetic orientation of magnetoactive material for connecting
beams of an array.
[0071] FIG. 20 is a schematic illustrating a method of
magnetic-field-assisted multi-material 3D printing for in-situ
magnetic programming of magnetoactive material.
DETAILED DESCRIPTION
[0072] A description of example embodiments follows.
[0073] As shown in FIG. 1, expandable arrays comprising a
shape-memory polymer can be made using 3D printing techniques,
including P.mu.SL techniques. In an expanded state, the receptacles
of the expandable array can be configured to interface with, or fit
within a multiwell plate. The array can be configured such that,
upon exposure to a stimulus, the shape-memory polymer reverts to a
contracted state that can fit within a container of a different
dimension, such as a histology cassette, as will be described
further below.
[0074] P.mu.SL is capable of fabricating complex 3D
micro-structures in a bottom-up, layer-by-layer fashion. Generally,
P.mu.SL techniques involve the following steps. A digital model
created by computer-aided-design (CAD) software is first sliced
into a series of closely spaced horizontal planes. These
two-dimensional slices are digitized as an image and transmitted to
a dynamic mask, which projects the image through a reduction lens
into a bath of photosensitive polymer resin. The exposed material
cures, and the stage on which it rests is lowered to repeat the
process with the next image slice. A schematic representation of
this process is shown in FIG. 1 at section (a).
[0075] P.mu.SL processes can rapidly generate complex 3D
geometries, for example, within minutes, with photo-curable
polymers. The high resolution (<5 .mu.m) offered by P.mu.SL is
at least an order of magnitude better than most 3DP techniques.
Scalability is another prominent attribute of P.mu.SL over other
existing 3D printing techniques. Unlike other widely used 3DP
processes where a time-consuming raster scanning of a laser beam or
an injection nozzle must be performed for each single layer (a
serial process), P.mu.SL solidifies the entire layer with a single
illumination of ultra-violet (UV) light within a few seconds (a
parallel process). Therefore, fabrication of a complex 3D structure
could be completed within an hour, compared to the lengthy
processing time of several hours to days for other 3D printing
methods. Also, by adopting step-and-repeat process, the build-area
of P.mu.SL can be extended to a larger area without compromising
resolution.
[0076] Furthermore, being able to modulate UV light intensity
digitally and individually at a single pixel level, P.mu.SL
provides for the flexibility to generate the desired material
properties and refine their spatial distribution. The intensity of
the light exposure strongly influences the crosslinking density of
photo-polymerized material, which is an important factor in
determining and adjusting physical properties of a polymer, such as
elastic modulus, molecular permeability and swelling ratio.
Molecular diffusivity of the polymer can be adjusted to provide for
receptacles that allow for culture medium and growth factors to
diffuse across the receptacle wall.
[0077] Smart materials are materials that can actively deform and
reconfigure when exposed to external stimuli. 3D printing of
shape-shifting materials, such as stimuli-responsive hydrogels and
shape memory polymers, has been explored and is termed 4D printing,
with the 4th dimension being the time-dependent shape change of 3D
printed objects in response to an environmental stimulus [6-8].
[0078] 4D printing of programmable smart material can be used to
generate receptacles, and arrays of receptacles, for use in
processing biological samples, such as 3D cultures involving
cellular spheres and organoids, or tissue samples. Typically, such
biological samples are cultured in multi-well plates. Following
culturing, or tissue collection, the samples are transferred to a
histology cassette for further analysis by microscopy techniques.
The transfer of the biological samples from multiwell plates to a
histology cassette is time intensive and manually detailed, often
taking about four days and requiring multiple steps to preserve the
relative orientation of the samples.
[0079] Expandable arrays are described that can interface with or
fit within multiwell plates, or other
cell-culturing/tissue-collection vessels, and can advantageously
provide for streamlined transport of biological samples from the
multiwell plates to containers of a different dimension, such as
histology cassettes, upon completion of cell-culturing or tissue
collection. For example, an expandable array can be configured to
transition from a larger footprint (e.g., a 96-well plate) to a
smaller footprint (e.g., a paraffin embedding block), while
retaining the biological sample(s) in a relative orientation.
[0080] As used herein, the term "array" applies to any
configuration of two or more receptacles for receiving a biological
sample, with at least a subset of the receptacles connected to one
another by a beam. For example, the array can be a regularly shaped
or patterned array, such as an 8.times.12 array configured to
interface with a 96-well plate, or an irregularly shaped or
patterned array, such as an array of 3 receptacles arranged in a
triangle or 7 receptacles arranged in a circle.
[0081] The term "beam," as used herein, applies to any connecting
element extending between receptacles of an array. Beams can extend
between upper, lower, and side surfaces of receptacles, in any
combination. For example, a beam can be a corrugated or serpentine
connecting element extending between sides of receptacles. A beam
can also be a helical connecting element that extends from an upper
surface of a receptacle. Beams may be integral with the receptacles
of an array, integral with other beams, or integral with both
receptacles and other beams. Alternatively, beams can be coupled to
receptacles and/or to other beams, such as through bonding. Beams
may alternatively be referred to as bridges.
[0082] As used herein, the term "receptacle" applies to any
structure configured to retain a volume of a fluid or solid,
including, for example, cells, cell culture media, and tissue
samples. Receptacles may be alternatively referred to as
baskets.
[0083] An example of an expandable array 100, including a method of
making and using an expandable array, is shown in FIG. 1. As
illustrated, the array 100 is an 8.times.12 3D cell culture array,
which, as printed by P.mu.SL, has a dimension that fits a paraffin
embedding cassette 120 (e.g., 24 m.times.30 m.times.11 mm). See
(1), (2), and (6) of FIG. 1. The array 100 includes a plurality of
receptacles 102, illustrated as baskets, each basket having a
dimension allowing it to fit within a well of a 96-well plate
(e.g., a diameter of 1 mm, a wall thickness of 100 .mu.m, and a
U-shaped bottom). See (3) of FIG. 1. Each basket is connected to
neighboring baskets with a beam 104, alternatively referred to as a
bridge, such as a corrugated or serpentine beam. When the beams 104
are stretched, a center-to-center distance between the baskets
matches that of the wells of a multiwell plate, such as the
standard 96-well plate illustrated in FIG. 1. In particular, as
illustrated, a conversation ratio between the dimensions of the two
states (e.g., a contracted/printed state and an expanded state) can
be about 1:4-5 and can be provided by 4D printing. See (3), (4),
and (5) of FIG. 1.
[0084] As illustrated, the array is printed in the contracted, or
shrunken, configuration (as shown in steps (1)-(3) of FIG. 1) with
an original dimension that matches that of an interior of a
paraffin-embedding cassette. After printing, the array is
mechanically and bidirectionally stretched to the expanded, or
extended, configuration with a dimension that is about 4-5 times
that of the original dimension and matching that of a standard 96
well plate (as shown in step (3) of FIG. 1). Optionally, the array
can include handles, such as right and left handles 106, for ease
of manipulation and assistance in maintaining a correct
orientation. While a rectangular array is depicted in FIG. 1 with
the handles 106 disposed at opposing ends of the array of
receptacles, handles may be positioned at orientation for both
regularly and irregularly patterned arrays. The handles can be
connected to the receptacles 102, to the beams 104, or to both the
receptacles and the beams.
[0085] Since the basket array is printed with shape memory polymer,
its stretched dimension can be temporarily fixed in the extended
configuration. In the extended configuration, the array can be
transferred to a standard 96 well plate for 3D cell culturing
processes. During the cell culture period, the extended dimension
can be retained by itself without any additional aid. P.mu.SL
printing advantageously provides for a tunable molecular
diffusivity of the basket such that the basket can allow for
material exchange while the cell culture is retained inside each
basket. Once cell culturing is completed, the culture can then be
subjected to formalin fixation and, optionally, the plate can be
subjected to brief centrifugation to cause the spheres or organoids
to lie at a same level at the bottom of the baskets. The rounded
bottom of the baskets can help to maintain the shape of the spheres
and organoids during processing.
[0086] The entire array can be taken out of the 96 well plate and
placed in an incubator, or exposed to a temperature change. In the
case of a thermally responsive shape-memory polymer, the
temperature can be gradually increased to above the glass
transition temperature of the shape memory polymer, upon which the
basket array will return to its shrunken configuration. See (4) and
(5) of FIG. 1.
[0087] Another example of a cell culture array is shown in FIGS.
2A-2C in both expanded (FIG. 2B) and contracted (FIGS. 2A, 2C)
states. The array 200 includes four receptacles 202, each
receptacle including two beams 204. The beams 204 are shown in a
corrugated configuration, extending between sides of the
receptacles 202 to each neighboring receptacle. FIGS. 3A-3D
illustrate contraction of the cell culture array 200 from an
expanded configuration (FIG. 3A) through intermediary
configurations (FIGS. 3B-C) to a shrunken configuration (FIG. 3D),
each basket of the array having within it a liquid, which, as shown
in the figures, does not spill as the array moves from the expanded
state to the contracted state.
[0088] Mechanical transformation of the array can occur mostly on
the connecting elements, or beams, rather than on the basket
itself, so mechanical perturbation or disturbance to the culture
inside each basket is minimal. In this process, the geometric
expansion is achieved by stretching of the connecting members
located between baskets (not the baskets themselves). In such a
configuration, with little or no deformation in z-direction during
a shape programming process (e.g., mechanical extension to the
expanded state), there is likewise little to no contortion in the z
plane during the shape recovery process (e.g., thermally-induced
contraction to the contracted state).
[0089] The receptacles of an array can be formed of a same
shape-memory polymer as that of the connecting members during the
P.mu.SL printing process. However, the baskets may alternatively be
formed of a different material than that of the connecting members,
including for example, a non-shape-memory material.
[0090] Furthermore, as illustrated, each basket is connected to
each neighboring basket, with the internal baskets of the array of
FIG. 1 each connected to each of its four neighboring baskets. In
such a configuration, the mechanical force applied to each basket
is symmetric, which can help to keep the basket in an upright
orientation during expansion and contraction. However, the internal
baskets of an array can be connected to fewer neighboring baskets,
such alternating rows of missing beams in an x- or y-direction, or
in the case of an irregularly shaped array, internal baskets may
each have more than four neighboring baskets and, as such, more
than four beams may be connected to each internal basked.
[0091] The transition temperature of the shape memory polymer can
be tuned to a temperature that is (i) above the 37.degree. C. cell
culture temperature so that the basket array can retain its
extended dimension during 3D cell culture and (ii)
.ltoreq.50.degree. C. or .ltoreq.80.degree. C. as may be needed to
prevent any thermal damage to the cell culture or tissue sample.
Once the array returns to its originally printed/contracted
configuration it can then be transferred to a paraffin-embedding
histology cassette for subsequent fixation and paraffin-embedding
processes. See (5)-(6) of FIG. 1.
[0092] Furthermore, a stiffness of the shape memory polymer can be
tuned during the P.mu.SL process such that it can be easily cut and
sliced with a microtome after the paraffin embedding process.
[0093] The polymer can be an acrylate-based or methacrylate-based
shape memory polymer. Such polymers advantageously provide for
tunability in terms of elastic modulus, extent of deformation, and
sensitivity to a stimulus that triggers glassy-rubbery transition.
Chemical and thermo-mechanical characterization of the polymers can
be assessed by Differential Scanning calorimetry (DSC), Fourier
Transform Infrared (FTIR) spectroscopy, and/or Dynamic Mechanical
Analysis (DMA) to ascertain an optimal combination of materials
properties.
[0094] Preliminary results are shown in FIGS. 4A-4C for a
shape-memory polymer (SMP) or varying composition. As shown in
FIGS. 4A-4C, an elasticity of the SMP changes up to 3 orders of
magnitude with a mild temperature change between 20.degree. C. to
80.degree. C. The effects of chemical composition, molecular weight
of polymer, photo-initiator concentration and cross-linking density
on the material's behaviors such as stiffness, strength, toughness,
energy absorption, and glass transition temperature can be
optimized to obtain desired material properties. The rubbery
modulus E.sub.r of the polymer increases with an increase in
crosslinking density as expected from entropic elasticity,
E.sub.r=(3.rho.RT)/M.sub.c; where, R is the gas constant, T is
absolute temperature, .rho. is polymer density, and M.sub.c is
average molecular weight between crosslinks. The ratio
.rho./M.sub.c is the crosslinking density of the polymer network
and can be obtained from a photo-polymerization model. Also, the
Couchman equation [9] can be used to predict the glass transition
temperature T.sub.g of the cured SMP from the prescribed ratio of
SMP and crosslinker:
1/T=M.sub.1/T.sub.g.sup.1+M.sub.2/T.sub.g.sup.2; where
T.sub.g.sup.1 and T.sub.g.sup.2 are the glass transition
temperatures of the respective pure-components, and M.sub.1 and
M.sub.2 are the corresponding mass fractions of the SMP and
crosslinker, respectively.
[0095] Experimental techniques including Fourier transform infrared
spectroscopy (FTIR), Raman spectroscopy and Zetasizer can be used
to characterize conversion ratio and molecular weight of the
polymer. Experimental techniques including various microscopy,
rheometer, differential scanning calorimetry (DSC), dynamic
mechanical analysis (DMA), and thermo-mechanical analysis (TMA) can
be used to characterize and assess the performance of synthesized
materials. As such, a desired shape transformation at a desired
temperature above or below cell culture temperature can be provided
for an expandable array. Furthermore, a rubbery modulus can be
tuned to a low value such that the receptacles, or baskets, or the
array can be easily sectioned using microtomes. In addition, or
alternatively, the SMP can be selected or configured to dissolve or
degrade, for example, during histology processing.
[0096] Examples of suitable shape-memory polymers include
polyacrylic acid, polyethylene glycol diacrylate, polyethylene
glycol dimethacrylate, diethylene glycol dimethacrylate, bisphenol
A ethoxylate dimethacrylate, tert-butyl acrylate, and n-Butyl
methacrylate. The shape-memory polymer can be compatible with
P.mu.SL printing methods and can be tuned as described above.
[0097] As shown in FIGS. 1-3D, the beams connecting each of the
receptacles of the array have a serpentine shape. However, other
shapes are possible. For example, the shape can be pleated or
corrugated, or otherwise include an undulating shape that permits
both a lengthened and a contracted state. The number of folds,
pleats, or turns of each beam, as well as the length of each beam
can be adjusted to permit expansion and contraction to varying
lengths. As such, expandable arrays can be customized to interface
with multiwell plates or other cell-culturing/tissue collection
vessels of varying shapes and sizes. Placement of the beams with
respect to the receptacles can also be customized to accommodate
various cell-culturing and tissue collection vessels. For example,
for a multiwell plate, beams may be disposed to extend from an
upper surface of the receptacles to enable the receptacles to be
disposed within the wells without or with minimal beam
interference. In another example, beams may be disposed to extend
from a lower surface of the receptacles to accommodate a hanging
cell culture apparatus.
[0098] Another example of an expandable array is shown in FIGS.
5A-5C. The array 500 includes a plurality of receptacles 502, or
cell tubes, having beams 504 in a helical bridge configuration. The
helical bridges 504 are shown in a contracted state in FIG. 5B and
in an expanded state in FIG. 5C. The helical bridges 504 can
include a joining element 506 disposed at an end of each beam for
connection to a neighboring beam, for marker placement, or both.
Upon stretching of the array 500, the helical bridges 504 can
unwind to accommodate a dimensional change, as illustrated in FIG.
5A. When heated, the helical bridges 504 can revert to their
original shape.
[0099] The receptacle and beam configuration shown in FIGS. 5A-5C
can advantageously provide for larger-volume receptacles over the
configuration shown in FIG. 1 as the beams are disposed above,
rather than between, the receptacles in the contracted state.
[0100] For conventional, multiwell plates, such as a 96-well,
24-well, or 6-well plate, the expandable array can include a
plurality of receptacles that are arranged in, respectively, an
8.times.12, 4.times.6, or 2.times.3 array. For a conventional
histology cassette, the expandable array can have a width of about
20 mm to about 30 mm (e.g., 24 mm) and a length of about 25 mm to
about 35 mm (e.g., 30 mm) when each beam is in the contracted
state.
[0101] The receptacles of an expanded array can also vary to
interface with the size and shape of the intended
cell-culturing/tissue collection vessel. For a conventional
multiwell plate, for example, it may be desirable to have each
receptacle comprise a basket-like shape, with a bottom of each
basket located approximately 2 mm above a bottom of the plate well.
For such a conventional multiwell plate, each receptacle of an
expandable array can have a diameter of about 0.5 mm to about 1.5
mm (e.g., 1 mm), a depth of about 5 mm to about 15 mm (e.g., 11
mm), and a wall thickness of about 50 .mu.m to about 150 .mu.m
(e.g. 100 .mu.m).
[0102] Expandable arrays can be included within a kit that further
includes materials for cell-culturing, such as one or more
biomolecules (e.g., a growth factor, an extracellular matrix
component) and/or a cell culture medium or ingredients for making a
cell culture medium.
[0103] Stretching of expandable arrays can optionally be performed
by a stretching device, alternatively referred to as a stretcher.
An example of a stretching device is shown in FIGS. 9A and 9B, and
use of the device is shown in FIG. 8. A top plate of the stretcher
900 includes a plurality of straight rails 902; and, a bottom plate
of the stretcher 900 includes a plurality of curved rails 904
(shown in transparency in FIG. 9A). As illustrated, the patterns of
rails connect locations of distributed baskets 906 of dimensions A1
and B1 in a compact state and A2 and B2 in an expanded state. The
rails of the upper plate of the stretcher 900 can be of a width to
accommodate a diameter of a top portion of an expandable array, and
rails of the lower plate can be of a width to accommodate a
diameter of a bottom portion of an expandable array. Outer baskets
of the array sitting in both rails can move from a compact
configuration to an expanded configuration by rotating the top
plate against the bottom plate, as shown in FIG. 9B.
[0104] Expandable arrays can optionally be placed in a fixture
device prior to placement within a histology cassette, or other
vessel. An example of a fixture 800 is shown in FIGS. 10A and 10B.
The fixture can have dimensions A.times.C corresponding to widths
of a histology cassette, or other containing vessel in which an
expandable array is to be placed. A window 802 can be included in
the fixture for viewing of the array. The fixture can further
include pins 804 configured to align baskets disposed around an
edge of an array with edge wells of a multi-well plate. For
example, a distance B between pins can be about 9 mm, corresponding
to a distance between wells of a 96-well plate. Fixtures can
advantageously prohibit or restrain the SMD of the expandable array
from contracting to its compact state during cell culturing
processes.
[0105] Methods of making expandable cell culture arrays can include
P.mu.SL techniques, as shown in FIGS. 1 and 5A. In particular, the
method can include discretizing a 3D computer-aided design (CAD)
model of the receptacles and beams of an array and displaying the
discretized images on a digital micromirror (DMD.TM.) device to
apply a dynamic virtual photomask. UV light, such as illimitation
from a light emitting diode (LED), is reflected off the dynamic
mask and focused on a surface of a photocurable liquid polymer
through a projection lens to form a layer of the array. The arrays
can then be built, layer upon layer, by repeating the process under
all layers are completed. While P.mu.SL techniques are described in
the following Example, other additive manufacturing techniques or
molding techniques can be employed for manufacture of cell culture
arrays.
[0106] Methods of operating expandable cell culture arrays can
include uniformly stretching the arrays to the dimensions of a well
plate. The cell culture arrays can thereby be programmed to retain
the dimensions of the well plate. The programmed cell culture
arrays can then be transferred to the well plate with a one-to-one
matching of the receptacles of the array and the wells of the
plate. Cells, cell culture media, drug compositions, and other
materials, or any combination thereof, can then be placed within
the receptacles. For cell culturing, upon cell seeding within the
receptacles of the array, the well plate, including the array, can
be placed in an incubator or oven for cell culturing. To fix cells
within the receptacles, formalin can be introduced. The array can
then be removed from the well plate and heated to a shape recovery
temperature to cause the array to revert to a compact (e.g.,
printed) configuration. For histology processing the compact
configuration of the array can be of a dimension that fits within a
histology cassette. The array, including cell contents can then be
transferred to the histology cassette. Paraffin wax or other
material can then be introduced prior to sectioning. Sectioning can
be performed, such as with a microtome to obtain thin,
cross-sectional films for analysis.
[0107] While example arrays have been described as receiving
biological samples, expandable arrays may also be configured to
receive non-biological samples, and methods of using such
expandable arrays can include placing a non-biological sample
within the receptacles of the array.
[0108] Furthermore, while 3DP techniques have been described as
methods by which expandable arrays may be manufactured, molding
processes may instead be applied to create such expandable
arrays.
[0109] Expandable arrays can include other programmable materials
that enable connecting beams of the array to transition from an
expanded state to a contracted state. For example, the programmable
material can be a magnetoactive material. Magnetoactive materials
are materials that can be programmed to respond to magnetic fields,
such as with a large deformation or tunable mechanical properties.
Examples of magnetoactive materials include elastomers or other
polymers within which magnetic or magnetizable particles are
disposed. With connecting beams comprising a magnetoactive
material, the application or adjustment of a magnetic field can
provide a stimulus that initiates contraction of the array.
[0110] As shown in FIGS. 17 and 18A-18B, an array (such as arrays
100, 500) can include a plurality of receptacles 302 connected to
one another by beams 304 that comprise a magnetoactive material.
The beams 304 can include at least two sections 310a, 310b
programmed to have an opposite magnetic orientation. FIG. 18A
illustrates the connecting beams in an extended state, and FIG. 18B
illustrates the connecting beams in a contracted state. In an
extended state, the beams 304 extend substantially horizontally
between neighboring receptacles 302. Upon application of a magnetic
field, the connecting beams responsively fold into the contracted
state. The fold can occur at a location 312 at which the magnetic
orientation of the material transitions from that of section 310a
to that of section 310b.
[0111] While the example receptacles and beams of FIGS. 17 and
18A-18B are illustrated to provide for a single fold, additional
folds can be included. For example, the connecting beams can
include more than two sections of differing magnetic orientations.
Three, four, five, six or more sections can be programmed to have
differing or alternating magnetic field orientations, thereby
allowing the connecting beam to adjust to a corrugated shape upon
contraction. As illustrated, the connecting beam is programmed to
fold vertically; however, the beams can be configured to respond by
adjusting to other configurations. For example, the beams can
comprise the geometric structures shown in FIGS. 1 and 5, with
application of a magnetic field causing the beams to fold
horizontally or to migrate upwards to a more compact helical
configuration.
[0112] As illustrated, a magnetic field can be applied by, for
example, a magnet placed above or proximate to the array. The
programmed orientation of magnetic (e.g., ferromagnetic)
microparticles embedded within the elastomer or polymer material
can provide for transitions between substantially unfolded (e.g.,
flat) and substantially folded states, depending upon orientation
of the applied magnet. The transition between these states can be
reversible. As illustrated, an applied magnet can be flipped with
respect to the array such that it is either attracting or repelling
the magnetic microparticles, the magnetoactive material
responsively causing folding or unfolding of the connecting beams.
Once in the folded, or otherwise contracted, state, the array can
be transferred, for example, to a histology cassette, as described
above.
[0113] Structural features of arrays comprising magnetoactive
materials can be similar to those described herein with respect to
shape-memory polymers. In particular, the arrays can be of any
configuration (e.g., an 8.times.12, 4.times.6, or 2.times.3
configuration, a configuration with handle(s) disposed at a
perimeter of the array, etc.) with beams and receptacles of various
shapes, sizes, and dimensions (e.g., rounded-bottom receptacles,
mesh receptacles, etc.).
[0114] Methods of using arrays comprising magnetoactive materials
and methods of maintaining biological samples with such arrays are
also similar to those described herein with respect to shape-memory
polymers, differing in that the application of a stimulus includes
exposure of the array to a magnetic field in place of light or
heat.
[0115] An expandable array can provide for a direct transfer of a
large cell-culture array from a standard multi-well plate to a
histology cassette as a single specimen. The direct transfer can be
particularly helpful for organoid cultures. Organoids are
multi-cellular 3D cell cultures of stem cell-derived,
self-organizing miniature organs that replicate the key structural
and functional characteristics of their in vivo biology. Due to
their ability to emulate microarchitecture and functional
characteristics of native organs, organoids are emerging as a
promising approach for the modeling of development of various human
organs and pathologies. Microscopy is a powerful tool for the
analysis of organoids because it reveals the spatial arrangement
and biological heterogeneity within the organoid. However, it must
be preceded by histology sectioning that requires slow, laborious,
and mostly manual process of harvesting organoids, converting them
into histology specimens, embedding them in paraffin wax, slowly
sectioning through the specimen using a microtome to locate the
multi-cellular aggregates, and then staining to give contrast to
the tissue as well as highlighting particular features of interest.
In particular, when a microwell plate is employed for culturing and
assaying a large number of organoids for drug screening, a series
of repetitive histology sectioning for individual organoids
canimpede effective analysis. In addition to increasing labor costs
for histology specialists, the slow and serial nature of the
processing steps is also a major roadblock to rapid and effective
drug discovery for aggressive tumors such as glioblastoma.
[0116] Expandable arrays can significantly improve the time and
effort involved in processing organoid samples for histology. As
shown in FIGS. 1 and 17, at the end of a cell culture process. The
expandable array containing three-dimensional (3D) organoid
cultures can be removed from a multi-well plate as a single unit
and collapsed to a footprint approximately four-times smaller
(e.g., from dimensions of about 110 m.times.65 mm for a 96 well
plate to about 30 m.times.20 mm for a histology cassette). As a
result, the entire array of cultured organoids can be directly
transferred to a histology cassette as a single specimen while
preserving the registry and orientation of the organoids.
Subsequent paraffin embedding and histology sectioning can yield an
ordered array of organoid sections deposited onto a single
microscope slide. The processing of all 96 sections in parallel
eliminates the need for 96 repetitions of the routine histological
analysis processing steps, which can take several days to weeks for
an array of such size.
[0117] Expandable arrays comprising magnetoactive material can
provide for transition of the array through exposure to a magnetic
field, which can be provided by a handheld magnet and which does
not require particular lighting and heating equipment to transition
the array to its contracted state. The application of a magnetic
field can further provide for minimal, if any, influence on the
biological samples contained within the array receptacles. Magnetic
stimulation can provide for a fast, non-contact, and non-cytotoxic
stimulus for transition the array to its contracted state.
Furthermore, the receptacles of an expandable array can be formed
from a different material than the magnetoactive material
comprising the connecting beams, thereby providing for minimal
disturbance to the biological samples during transition of the
array to its contracted state as the receptacles remain
structurally unaffected by application of the magnetic field.
[0118] The expandable arrays can be created by multi-material
digital 3D printing techniques (e.g., projection
microstereolithography (P.mu.SL)). In particular a 3D printable
magnetoactive smart material can be synthesized, as shown in FIG.
19. A composite resin can be prepared using a photo-curable polymer
(e.g., poly(ethylene glycol) diacrylate, PEGDA), a photo-initiator
(e.g., Irgacure 819), and ferromagnetic microparticles (e.g.,
neodymium-iron-boron, NdFeB). The resin composition can be adjusted
to provide for a desired viscosity, dispersion, reactivity, and
magnetic sensitivity. Magnetic sensitivity can be measured or
confirmed by a rheometer, Fourier-transform infrared spectroscopy
(FTIR), and dynamic mechanical analysis. A UV curing testbed with a
variable magnetic field generator 330 can be used to provide
selective magnetization of a resin. As illustrated, the magnetic
field generator can include an electromagnet with at least two
degrees of freedom (DOF) for motion control. By changing field
direction and curing the selected region with UV, different areas
can be programmed to have different magnetization orientation,
resulting in a prescribed transformation with a magnetic field
(e.g., folding transformation as shown in FIG. 17).
[0119] As illustrated in FIG. 20, a P.mu.SL system can be modified
to include a variable magnetic field generator. Strength and
direction of a magnetic field can be controlled around the printing
chamber, where material can be readily changed as needed. Such a
system can provide for flexibility in printing an array comprising
multiple materials. For example, receptacles of the array can be
printed with a first material providing for biocompatibility while
connecting beams of the array are printed with a second,
magnetically-programmable material. Inclusion of an electromagnet
can provide for in-situ magnetic programming of the smart material.
Performance and functionality of a smart cell-culture array
comprising a magnetoactive material can be evaluated by real-time
by monitoring of the magnetic field and associated deformation. A
3D printed array can be soaked in acetone followed by an ethanol
wash and sterilization to remove residual photo-chemicals.
[0120] While a P.mu.SL system is shown and described, manufacture
of arrays comprising magnetoactive materials is not limited to such
systems. As material selection can be expanded over arrays
comprising shape-memory polymers, other manufacturing methods can
be employed. For example, the arrays can be formed by injection
molding, providing for improved scalability and higher throughput
over P.mu.SL techniques.
[0121] The receptacles can be formed with, for example, PEGDA,
which is biocompatible, permeable to culture medium, non-adherent
to cells, and 3D printable or moldable. Other suitable materials
for receptacles include 1,6-Hexanediol diacrylate (HDDA),
Polyacrylamide (PAAm), and Poly(2-hydroxyethyl methacrylate)
(pHEMA).
[0122] The connecting beams can be printed with a magnetoactive
material as described above. While the connecting beams may
comprise a same biocompatible polymer as provided for the
receptacles, other polymer or elastomer materials, including
non-biocompatible materials, can be used instead. The polymer or
elastomer material can be any material within which magnetic or
magnetizable structures can be embedded. Examples of suitable
polymer or elastomer materials include Polydimethylsiloxane (PDMS)
and Polyurethane (PU).
[0123] The magnetic or magnetizable structures can be ferromagnetic
or ferrimagnetic and can be in the form of particles, such as
microparticles. Examples of suitable magnetic materials include
neodymium-iron-boron (NdFeB), samarium cobalt (SmCo), alnico
(AlNiCo), ferrite (Fe.sub.3O.sub.4), and Chromium (IV) oxide
(CrO2). In another example, the magnetoactive material can comprise
a magnetic rubber, such as a synthetic rubber or polyvinyl chloride
(PVC) impregnated with a ferrite powder (e.g., barium,
strontium).
[0124] A concentration and size of magnetic particles embedded
within the polymer can vary to provide for an appropriate level of
material flexibility and level or response to a magnetic field
stimulus. In general, magnetic particles of smaller sizes can
provide for denser magnetic lattices within the polymer material
and, consequently, greater magnetic response. The magnetic
particles can be microparticles or nanoparticles. For example, the
magnetic particles can have diameters of about 0.5 .mu.m, 1 .mu.m,
5 .mu.m, 10 .mu.m, 25 .mu.m, or 50 .mu.m. A concentration of
magnetic particles within the polymer material can be about 1%, 5%,
10%, 15%, 20%, or 30% by volume. The particles can be monodispersed
throughout the polymer comprising the connecting beam.
Example 1
4D Cell-Culture Arrays
[0125] Expandable arrays were created for cell culturing, the
expandable arrays configured to transform between the size of a
histology cassette and the size of a 96-well plate (e.g.,
3.6.times. the size of the histology cassette) while maintaining a
same layout in both forms. Expandable arrays were manufactured and
operated according to the procedure shown in FIG. 5A.
[0126] Projection Micro-Stereolithography (P.mu.SL)
[0127] P.mu.SL techniques were employed for the manufacture of the
cell-culture arrays. The resolution of the digital dynamic mask was
1920.times.1080 and the projection area was 24.times.14 mm,
providing for a nominal resolution of 13 .mu.m. A resolution of
800.times.800 (.about.10 m.times.10 mm) was used in printing to
ensure high uniformity in light intensity. To print full basket
arrays with a dimension of 30 m.times.20 m.times.11.2 mm, a 3-by-2
stitching of projections within one layer was employed (horizontal
movement of printed structure using XY stages).
[0128] A custom-built P.mu.SL system was used in this work. It
consisted of a UV LED (365 nm) (L10561,Hamamatsu), a collimating
lens (LBF254-150, Thorlabs), a digital micro-mirror device (DMDTM)
(DLPLCR6500EVM, Texas Instruments), three motorized linear stages
(MTS50-Z8, Thorlabs), and a projection lens (Thorlabs). Printing
parameters we used include a light intensity of 29 mW cm-2, a layer
thickness of 50 and a curing time of 1 s. The entire P.mu.SL system
was kept in a UV blocking enclosure.
[0129] Shape Memory Polymer (SMP) Materials
[0130] Shape memory polymer (SMP) was included as a constituent
material of the 3D cell-culture basket arrays to enable
transformation between configurations.
[0131] All chemicals, including liquid oligomers, photoinitiator
(PI), and photo absorber (PA), were purchased from Sigma-Aldrich
(St. Louis, Mo., USA) and used as received. Poly(ethyleneglycol)
diacrylate (PEGDA) (Mn250) and Bisphenol A ethoxylate
dimethacrylate (BPA) (Mn1700) were mixed at a ratio of 9:1 in
weight. Phenylbis(2,4,6-trimethylbenzoyl) phosphine and Sudan I
were added at the concentration of 2 wt. % and 0.1 wt. % of the
precursor solution as PI and PA, respectively.
[0132] Post Processing
[0133] After printing, the arrays were treated using
post-processing procedures prior to cell culturing processes.
[0134] Printed structures were rinsed in fresh ethanol for 30 s for
3 times to remove any uncured precursor solution. After being dried
in air until the absorbed ethanol evaporated, the structures were
rinsed in pentane one more times to avoid adhesion between bridges
and baskets. After pentane drying, the structure were post-cured in
a UV oven (CL-1000L, UVP, 365 nm) for 2 hours to polymerize all
unreacted ethyl group in acrylate/methacrylate . To eliminate
toxicity in remained PI and PA, fully crosslinked structures were
stored in an Acetone bath for 5 days. Structures taken out from the
Acetone bath were rinsed in ethanol one more time for sanitization
and were dried overnight at room temperature.
[0135] Dynamic Mechanical Analysis and Failure Strain
[0136] To characterize the SMP's thermomechanical properties, a
photocurable precursor solution was prepared using Poly(ethylene
glycol) diacrylate (PEGDA) and bisphenol A ethoxylate
dimethacrylate (Mn-1700) (BPA). Upon photo-polymerization, a
cross-linked polymer network is formed with these two materials. It
has been shown that a glass transition temperature T.sub.g can be
tailored by using different ratios of monomer and crosslinker. To
maintain shape fixity at 25.degree. C. (e.g., room temperature) and
trigger shape recovery at 50.degree. C. (e.g., an accepted maximum
temperature for cell viability), the SMP was designed to have a
weight ratio between PEGDA and BPA of 9:1. Thermomechanical
properties of the SMP were then characterized by dynamic mechanical
analysis (DMA) tests on both 3D printed and molded specimens.
[0137] For molded samples, an SMP precursor solution without PA was
injected into a mold of two glass slides separated by 1 mm spacers.
Glass slides were cleaned with ethanol and coated with RainX for
easy demolding. The precursor solution in the mold was cured in a
UV oven (CL-1000L, UVP, 365 nm) with a light intensity of 5 mW cm-2
for 20 min, yielding a fully crosslinked polymer film with a
thickness of 1 mm. Samples were laser cut to 40 m.times.8
mm.times.lmm rectangular specimens. For 3D printed samples, the
same printing parameters and post-processing procedure (except
toxicity-eliminating steps) were used. Dimensions of 3D printed
samples were 25 m.times.8 m.times.1 mm. DMA was conducted on a
dynamic mechanical analyzer (Q800, TA Instruments) using a tensile
loading mode. Testing parameters for DMA included strain of 0.2%,
frequency of 1 Hz, preload of 0.001 N, and force track of 150%.
Specimens were heated at 25.degree. C. for 10 min prior to each
test. Storage modulus, loss modulus, and tan .delta. were measured
as a function of temperature while temperature was increased to
75.degree. C. at a rate of 1.degree. C. min.sup.-1.
[0138] The results from DMA tests on both specimens are shown in
FIG. 6A. Note that the storage modulus of the SMP changes from 984
MPa at 25.degree. C. to 132 MPa at 75.degree. C. Tan .delta.
indicates that the SMP has a T.sub.g of 61.degree. C. Though
T.sub.g is higher than a maximum heating temperature of 50.degree.
C., recovery behavior was observed at 50.degree. C. and high
T.sub.g is desired to for better shape fixity at room
temperature.
[0139] For temperature dependent failure strain tests, molded films
were made using the same protocol from the DMA test. The molded
films were laser cut into a dog-bone shape (gauge section:
16.5.times.3.times.1 mm) to measure strain at failure of material
at different temperatures. Two grippers clamped on two ends of
rectangular specimens. An air chamber with Peltier heater
(CP-061HT, Technology, Inc.) underneath was used to control
temperature inside and a thermocouple connected to an NI
temperature module on cDAQ (NI 9171 and NI 9211, National
Instrument) was used to measure temperature. Two dots were marked
in the gauge section of dog-bone specimens and a digital camera
(Canon 60D) were set on top to monitor distance between dots. One
gripper was then manually moved at an average speed of 0.2%
sec.sup.-1 to stretch the sample until failure. Strain at failure
was then calculated using final distance divided by initial
distance between two dots.
[0140] Using the molded specimens that were laser cut into dog-bone
shape, stretchability of the SMP was tested by tensile test at four
different temperatures, the results of which are shown in FIG. 6B.
During basket arrays' transformation from histology cassette
configuration to 96-well plate configuration, a global dimensional
change of 3.6 times was required. Adequate stretchability can be an
important design constraint for limiting local strain to avoid
breakage during transformation. Four different temperatures between
25.degree. C. to 50.degree. C. were tested. Average stretchability
at each tested temperature varied from 12% to 14%, and minimum
stretchability among all measurements was slightly above 10%. The
result indicates local deformation during shape transformation
should be limited within 10% of strain.
[0141] To demonstrate SME, shape programming and shape recovery of
a 3D printed SMP beam was performed, the results of which are shown
in FIG. 6C. Stress, strain and temperature during the process are
plotted. The beam was compressed by 5% at 25.degree. C. While
maintaining the strain, the beam was heated to 50.degree. C. and
then cooled down to 25.degree. C. again. Note that required stress
to maintain the compressive strain reduced significantly due to
fixing of deformed shape. After removal of mechanical loading, the
deformed strain was retained at 3%. Even though only 60% of strain
was fixed after release, it can be further improved using longer
holding time and higher heating temperature. Upon heating back to
50.degree. C., the original height of the beam was completely
recovered.
[0142] Array Design
[0143] Arrays were designed as shown in FIGS. 5A-5C to meet
dimensional requirements for transport between a 96-well plate and
a histology cassette, as well as to satisfy stretchability and cell
culture requirements. To fit 12.times.8 basket arrays in the
histology cassette, a maximum dimension of one basket unit was
restrained to 2.5 m.times.2.5 m.times.11.2 mm. As shown in FIG. 7A,
the height of the cell tube was 3.2 mm and the height of the
helical bridge was 8 mm. The opening of the cell tube was designed
to be 1.8 mm in diameter to accommodate a 20 .mu.L pipette tip used
in cell seeding.
[0144] Wall thickness of cell tube and thickness of helical bridge
were 200 .mu.m. Width of helical bridge was 1.45 mm. Total length
after full extension of helical bridge without considering
constraint in local strain can be 22.1 mm. In the 96-well plate
configuration, each basket was to be stretched to 9 m.times.9 mm.
Height was approximately 10 mm due to unwinding of helical bridges.
Results from a numerical simulation with proper constraints of a
single unit basket revealed that local strain after stretching to
the 96-well plate configuration was lower than 5.1%, which is half
of the smallest measured failure strain from the experiments
described with respect to FIG. 6B and which indicated that no
breakage would occur during transformation. The receptacle and beam
design and the resulting numerical analysis of the design are shown
in FIG. 7A.
[0145] Cassette and well-plate configurations of the arrays are
shown in FIG. 7B. Assembly of the basket arrays consisted of unit
baskets having alternatively clockwise and counterclockwise helical
bridges. Based on trials, such a configuration demonstrated less
twisting of helical bridges during stretching than configurations
that included only one rotational direction. In the cassette
configuration, basket arrays were designed to have an overall
dimension of 30 m.times.20 m.times.11.2 mm. In a stretched,
well-plate configuration, the basket arrays were designed to have
an overall dimension of 101 m.times.65 m.times.10 mm, as shown in
FIG. 7B. Edge baskets were intentionally printed without bottoms
for fixing onto a well plate during cell culturing (see Array
Operation).
[0146] Results of heated recovery testing of the designed arrays
are shown in FIG. 7C. Heated recovery was measured quantitatively
using a customized digital image tracking code. Markers were
labeled on the connecting points between bridges and were traced
using digital image processing. D.sub.0 indicates original size of
a unit basket, which was 2.5 mm. D indicates measured size at
timestamps during heating. Heating started from room temperature at
0 min for capturing size after shape fixing. Once the temperature
reached 50.degree. C., the temperature was maintained until the end
of experiment. The starting ratio of D.sub.0/D at 0 min was 2.9
instead of 3.6 (ratio of size of well plate/size of cassette). This
was caused by imperfect shape fixing of SMP during shape
programming. After 120 min of heating, the basket arrays recovered
to 1.2 times its original size instead of 1. This was due to
friction between the bottom surface and basket arrays. From FIG.
7C, it can be seen that recovery mostly happened during the first
20 min of heating and slowed down significantly in the rest of
experiment. A 10-min heating time was chosen in further
cell-culturing operations as the temperature used for recovery was
maintained at 50.degree. C. (without heating from room temperature,
as in this characterization experiment). Since basket arrays are
flexible and can be squeezed to fit into cassettes after recovery,
100% recovery using long-time heating is not necessary and may be
undesirable as cells may be negatively affected by exposure to the
higher temperature of 50.degree. C. than 37.degree. C. for an
extended period of time.
[0147] For shape recovery characterization, black markers were
drawn on connecting parts of helical bridges, as shown in FIG. 11A.
A 45-degree mirror was used on top of basket arrays so a digital
camera (Canon 60D) can view the arrays from a horizontal
configuration. The basket arrays and mirror were kept inside a
temperature oven and the camera was maintained outside the oven. A
transparent window at the door of oven was used for visibility
during experiment. After obtaining video of shape recovery, an
example of which is shown in FIG. 11B, image processing using
MATLAB was applied on images at different timestamps to obtain
coordinates of markers. Detected markers were indicated using red
squares, as shown in FIG. 11C. Distance was then calculated to
between corresponding markers to obtain size of baskets. Since
there are clockwise (CW) and counterclockwise (CCW) baskets,
periodicity in a basket arrays was considered to be a combination
of one CW and one CCW basket. Six pairs of markers from a repeating
unit (two baskets) were measured at each timestamp. Then results
were divided by two to obtain average and standard deviation of
size of one basket.
[0148] Array Operation
[0149] Operation of the arrays is shown in FIG. 8. At room
temperature, a basket array in a cassette configuration was first
mounted on a customized stretcher, as shown in FIGS. 9A-9D. The
stretcher included eight rails configured to simultaneously move
all carriages sitting in the rails between the dimension of a
cassette and the dimension of a 96-well plate. To provide for
uniform stretching, a number of rails is desired to be the same as
the number of edge baskets. However, due to limited space at a
cassette configuration, only eight rails were fit in the test
device.
[0150] The top acrylic plate was laser cut with eight straight
rails. Patterns of rails connected locations of eight evenly
distributed baskets in the original configuration (17.5.times.27.5
mm) with locations of same baskets in the stretched configuration
(105.times.165 mm) (stretching capability of 6 times). A bottom
acrylic plate was laser cut with eight curved rails that are
compatible with straight rails. Top rails had a width of 5 mm and
bottom rails had a width of 3 mm. Cylindrical carriages had a
diameter of 5 mm in top portion and 3 mm in bottom portion.
Carriages with 12 needle pins (diameter of 0.8 mm) sitting in both
rails can move from a small configuration to a large configuration
by rotating the top plate against bottom plate.
[0151] The basket array was stretched to a 96-well plate
configuration by rotating the top and bottom plates against each
other at room temperature. The helical bridges of each basket
unwinded during rotation of the stretcher, as shown in FIGS. 9B and
9D. In the test device, in the compact configuration shown in FIGS.
9A and 9C, a first distance in the x-direction (A1) was 27.5 mm and
a first distance in the y-direction (B1) was 17.5. After rotation,
in the expanded configuration shown in FIGS. 9B and 9D, a second
distance in the x-direction (A2) was 165 mm and a second distance
in the y-direction (B2) was 105 mm.
[0152] After rotation, both the basket array and stretcher were
placed in a temperature oven at 50.degree. C. for 10 min and then
cooled down to room temperature to fix the stretched shape. Then
basket arrays were then removed from the stretcher with the
temporarily programmed shape.
[0153] At this stage, the temporary shape did not match exactly
with 96-well plate. Basket arrays were then mounted onto a fixture,
a schematic of which is shown in FIGS. 10A-10B, by slightly further
stretching of the edge baskets to match corresponding pins of the
fixture. The fixture was designed to match edge units of basket
arrays and a 96-well plate.
[0154] The fixture was 3D printed using a fused deposition modeling
(FDM) printer (grint, Stratasys). The fixture included a window in
its center and pins that match edge baskets with edge wells in
96-well plate. A CAD design of fixture the fixture is shown in
FIGS. 10A-B. The dimensions of the fixture, with reference to FIG.
10A were as follows: 99 mm (A), 9 mm (B), 64 mm (C).
[0155] Another advantage of including a fixture is to restrain the
SMPs recovery behavior over time. After fixing, a SMP will
gradually restore its original shape at a temperature dependent
speed (e.g., higher rate at higher temperature). Since cell
culturing processes typically occur at a temperature of 37.degree.
C. for two weeks, a fixture can ensure that shape recovery of the
array does not occur during this period of time.
[0156] The fixture with the basket array was then placed on a
96-well plate for cell seeding. Cells were injected into each
basket using micropipette and cell culture media were added into
wells and baskets. After cell culture, basket arrays were removed
from the fixture and heated to 50.degree. C. to induce shape
recovery. Once the array reached a cassette configuration, it was
ready for histology processing.
Example 2
Biocompatibility Verification of 4D Cell-Culture Arrays
[0157] Organoid growth in manufactured cell culture arrays was
examined to verify biocompatibility of the arrays. In particular,
3D-printed cell-culture arrays were fabricated as described in
Example 1 and used for histological analysis of patient derived
organoids (PDOs) for glioblastoma (GBM) therapy.
[0158] The biocompatibility of the basket arrays for generating GBM
spheres and GBM organoids and histological processing and imaging
was examined.
[0159] Sphere and organoid numbers, viability, and differentiation
potential were quantified upon basket memory reconfiguration at
50.degree. C. Use of the cell-culture array was shown to reduce
tissue fixation time from, historically, 1-3 days to 6 hours, as
shown in the histological processing steps shown in FIG. 12A and
obtained microtome sections shown in FIG. 12B. SMP baskets were
also determined to be compatible with automated processing methods.
Three rounds of histological processing and Hematoxylin and Eosin
(H&E) staining determined the processing time and cutting
parameters for use with the biomaterial, results of which are shown
in FIG. 12B.
[0160] SMP compatibility with 10% neutral buffered formalin
fixation was supported, while GBM cell integrity was maintained in
the twelve-step histological assay process shown in FIG. 12A.
Individual baskets, intact or sectioned, were used to facilitate
cell seeding and organoid formation, as shown in FIG. 13.
Cross-section, side-section, and magnified areas of sample baskets
without (top views) and with (bottom and right-side magnified
views) cells plated as organoids are shown in FIG. 13. Note that
the baskets allowed initiations of GBM PDOs within 72 hours, as
shown in the magnified view in FIG. 13.
[0161] While SMP components were compatible, PEGDA 700 developed
opacity with prolonged fixation and was replaced with PEGDA 250 in
the prototype basket arrays.
[0162] The effects of SMP components on cell viability were
examined in both U87 and primary GBM 3D cultures. Formation of U87
GBM spheres within one week was overall comparable with or without
SMP baskets, as shown in FIG. 14A. Bright field images of GBM
spheres derived from U87 cells in the presence of FBS and grown
with no basket and with baskets are shown in the bright field
images of FIG. 14A.
[0163] When primary GBM#50 cells were grown in either serum-free
sphere conditions (no matrigel) or as GBM organoids, large GBM
spheres and diversified organoids with multicellular connections
were detected after one or two weeks, respectively, in the absence
of basket arrays. With the basket arrays, the number and size of
primary spheres or organoids were significantly reduced (FIGS.
14B-C). GBM organoids grown in serum-free conditions with growth
factors derived from GBM#50 cells with no basket and with baskets
are shown in the bright field images of FIG. 14B. GBM #50 cells
grown in matrigel spheres with no basket and with baskets are shown
in the bright field images of FIG. 14C.
[0164] Unexpectedly, these studies suggested that serum or matrigel
could have neutralizing effects on the biomaterial components. To
investigate each component, it was first determined, by measuring
media levels in prolonged cultures, that baskets were not absorbing
media and, thus, were limiting growth factor availability. Notably,
prolonged culture media were yellow-tinted and more alkaline
compared to control culture, suggesting that the basket biomaterial
could be leaching low levels of chemicals that may interfere with
long-term organoid cultures.
[0165] SMP components, including poly (Ethylene glycol) diacrylate
250 (PEGDA 250), Bisphenol A (BPA), photo-initiator (PI) and
photo-activator (PA) were each examined in the GBM intracellular
ATP cell viability assay. Only PEGDA250, when used at three log
concentration of median dose (1,000 fold in excess of EC50 at 7.2
.mu.M) showed a significant loss of cell viability (FIGS. 15A-C),
suggesting that low PEGDA 250 levels may be leaching from the
basket during long-term organoid culture. Preincubation of the
basket array after 3D printing into culture media, PBS, 10% BSA, or
.beta.-mercaptoethanol (at 10 .mu.M or 50 .mu.M) did not reverse
the cell loss phenotype, but presoaking of basket arrays after 3D
printing into 100% acetone, followed by PBS and ethanol washes did
(FIGS. 16A-F). Based on these data, 3D printed SMP basket arrays
were established for PDO generation and drug assays to identify
effective treatments for primary GBM.
[0166] FIG. 15A illustrates the results of the cell titer glo assay
that utilizes ATP levels and was used to determine GBM viability in
the presence of component biomaterials. FIGS. 15B and 15C are
low-power images of bright fields of spheres cultured for one week
in the presence of the indicated chemicals and concentrations. The
insets are higher power (20.times.) magnification of the 5.times.
images. Only PEGDA250 when used at 7.2 .mu.M induced a significant
loss of cell viability.
[0167] Acetone soaking was shown to allow biomaterial basket GBM
sphere and organoid long-term culture, as shown in FIGS. 16A-F.
Different components of basket biomaterial were individually
examined at the final concentrations used in the prototype basket
array were shown to allow normal sphere culture (FIG. 16B). Soaking
of a basket array in a 100% acetone bath after 3D printing allowed
normal sphere culture (FIG. 16C). GBM organoid culture upon soaking
for 12-14 h in acetone gradually improved effects (FIG. 16D and
16F). The soaking of a basket array in a 100% acetone bath after 3D
printing allowed normal GBM organoid culture (FIG. 16E).
[0168] The platform developed was then examined with GBM tissues
for both paraffin embedding for histological analysis and genomic
sequencing, and with live GBM tissue for generating spheres and
organoids for drug sensitivity testing. GBM tissues were subjected
to exome sequencing to simultaneously detect the genetic
alterations characteristic for adult GBM (GlioSeq) and identify
deregulated pathways to guide the selection of targeted therapies.
GlioSeq analyzes 30 genes for single nucleotide variants (SNVs) and
indels, 24 genes for copy number variations (CNVs), and 14 types of
structural alterations in BRAF, EGFR, and FGFR3 genes in a single
workflow. Single cells were seeded at clonal densities in ultra-low
attachment plates with basket arrays for sphere formation or in
extracellular matrix droplets for organoid formation. GBM spheres
or organoids were kept in serum-free growth factor supplemented
conditions. The sphere assay is a functional assay to study GICs
expressing stemness factors such as NESTIN, SOX2, OLIG2 and ZEB129.
When bFGF and EGF were removed or GBM spheres cultured on
polyornithine coated-surfaces, GBM cells underwent differentiation
with GIC loss. In contrast, 3D cultured GBM organoids were
heterogenous and capable of interconnecting (mimicking brain cells)
and differentiating into cells with multiple cell phenotypes.
Immunofluorescence (IF) for the neural stem cell protein NESTIN,
and primitive neuroepithelium neuron-specific TUBULIN-beta-III and
mature astrocytic Glial fibrillary acidic protein allowed to
distinguish stemness from differentiation.
[0169] The basket arrays were used to deploy rapid single cell
derived sphere and organoid assays to assess tumor cell viability,
tumor invasion, terminal differentiation and resistance to therapy
for cancer drug discovery and drug validation. Single and/or clonal
GBM cell derived PDOs formed in 2 weeks and demonstrated invasion
of the semisolid matrix by extended invadopodia. PDOs were treated
for 72-hours with standard chemotherapy (TMZ) and/or molecularly
targeted agents, targeting mTOR, PI3K, BMI1, EGFR, and DDR, among
others. Following treatments, the entire 4D printed basket arrays
were evolved, with a 10-min heating step at 50.degree. C., to their
programmable cassette size to directly perform histological and IHC
validation on the same day, and with the convenience of maintaining
the same tissue plate arrangement. The concentrations inhibiting
viability by 50% (GI50), real time activated caspase 3 for
detection of apoptotic cells and GBM tumor cell invasion in live
intact organoid cells were less impacted by standard TMZ than
targeted therapies. Critically, treatment with molecularly targeted
agents alone or in combination had significantly more GBM organoid
cell killing than TMZ, particularly in apparently TMZ resistant
organoids, with targeted therapy reducing EGFR expression in
organoid cells that were not affected by TMZ treatment, and with
effective biomarker responses to targeted therapies, even at lower
level combinations.
[0170] The cell-culture array platform allowed the entire patient
tissue and drug response assessment to be completed in <20 days.
When including exome and/or single cell sequencing, histological,
IHC and targeted therapeutic assays, the array platform was
demonstrated to offer dynamic, automated and quantitative drug
analyses, thus allowing the discovery of novel preclinical
therapeutic approaches that can be assessed in clinical trials and
may be used to examine and select personalized therapies in
precision medicine oncology.
[0171] While example embodiments have been particularly shown and
described, it will be understood by those skilled in the art that
various changes in form and details may be made therein without
departing from the scope of the embodiments encompassed by the
appended claims.
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